Cloning the complete guinea pig cytomegalovirus genome as an infectious bacterial artificial chromosome with excisable origin of replication

Xiaohong Cui, Alistair McGregor, Mark R. Schleiss, Michael A. McVoy

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Congenital human cytomegalovirus infections are the major infectious cause of birth defects in the United States. How this virus crosses the placenta and causes fetal disease is poorly understood. Guinea pig cytomegalovirus (GPCMV) is a related virus that provides an important model for studying cytomegaloviral congenital transmission and pathogenesis. In order to facilitate genetic analysis of GPCMV, the 232 kb GPCMV genome was cloned as an infectious bacterial artificial chromosome (BAC). The BAC vector sequences were flanked by LoxP sites to allow efficient excision using Cre recombinase. All initial clones contained spontaneous deletions of viral sequences and reconstituted mutant viruses with impaired growth kinetics in vitro. The deletions in one BAC were repaired using Escherichia coli genetics. The resulting repaired BAC reconstituted a virus with in vitro replication kinetics identical to the wild type parental virus; moreover, its genome was indistinguishable from that of the wild type parental virus by restriction pattern analysis using multiple restriction enzymes. These results suggest that the repaired BAC is an authentic representation of the complete GPCMV genome. It should provide a valuable tool for evaluating the impact of genetic modifications on the safety and efficacy of live attenuated vaccines and for identifying genes important for congenital transmission and fetal disease.

Original languageEnglish (US)
Pages (from-to)231-239
Number of pages9
JournalJournal of Virological Methods
Issue number2
StatePublished - May 2008

Bibliographical note

Funding Information:
The authors are grateful to Gabriele Hahn and Wolfram Brune for providing pCre, Dong Yu for providing pYD-Tn1721 and very helpful advice on BAC cloning, and Greg Smith for providing pGS284, E. coli strain GS500, and very helpful advice on allelic exchange. This work was supported by a grant R01HD044864 from the National Institutes of Health.


  • BAC
  • Congenital infection
  • Cytomegalovirus


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